The invention relates to biologically active extracts obtained from the plant Salvadora persica Linneaus 1753. The invention also provides a process for obtaining the crude extract. Further, the invention provides pharmaceutical composition exhibiting biological activity, especially tocolytic activity.
The associated mangrove plants, Salvadora persica Linneaus 1753, belong to the order Salvadoracea and are shrubs or small trees with white flowers frequent in degraded mangrove swamps and saline banks all over the west coast of India. Large numbers of marine plants have been examined for bioactive substances. Nazarine et al., Indian Journal of Marine Sciences 27:499-501 (1998) have reported promising pharmacological activities in marine organisms from Indian waters.
There are several patents available from all over the world related to processes and compounds from nature for various purposes. Kwak et al. patented a process of extracting and purifying biologically effective ingredients from combined medicinal plants, along with the extract compositions (U.S. Pat. No.5,910,307 issued on Jun. 8, 1999). D""Amelio et al. disclosed therapeutic compositions and methods for treating skin using an extract from the Centipeda cunninghami plant (U.S. Pat. No. 5,804,206 issued on Sep. 8, 1998). Zimmerman et al. isolated compounds from the methanolic extract of the eelgrass Zostrea marina having significant antifouling aquatic properties (U.S. Pat. No. 5,607,741 issued on Mar. 4, 1997).
Betulinic acid, which is prepared from the compound betulin, has many pharmaceutical potentials. Pezzuto et al. disclosed methods of manufacturing betulinic acid from betulin (U.S. Pat. No. 5,804,575 issued on Sep. 8, 1998). The betulinic acid has been intensively investigated as a potential therapeutic agent for a variety of diseases (U.S. Pat. No. 5,962,527 issued Oct. 5, 1999). Pisha et al., in Nature Medicine 1:1046-1051 (1995), disclosed that betulinic acid has an unexpected selective anti-tumour activity against human melanoma, e.g., MEL-1, MEL-2 and MEL-4. In addition, Fujioka et al., J Nat. Prod. 57(2):243-247 (1994), disclosed that betulinic acid has anti-HIV activity in H9 lymphocytic cells. These inventors mentioned that the research directed to betulinic acid as a therapeutic agent is hindered because the betulinic acid is available in very limited quantities and at a very high cost. Ramadoss et al., in U.S. Pat. No. 6,048,847, which issued on Apr. 11, 2000, describes uses of betulinic acid and its derivatives for inhibiting cancer growth and a method of monitoring this. Kang et al. disclosed flavoured product additives in U.S. Pat. No. 5,948,460 issued on Sep. 7, 1999 in which ursolic acid was one of the compounds amongst a group of three compounds that was added to a flavoured product to reduce aftertaste in the product and enhance its sweetness like in a diet drink. It was also used as a constituent in a preparation for inhibition of skin Tumorigenesis.
Herman (U.S. Pat. No. 5,190,979 issued on Mar. 2, 1993) disclosed lupeol also as a compound that can make pharmacologically active terpene ozonides which have medicinal value.
The main object of the present invention is to provide a process for obtaining a crude extract from stem, leaves, and flowers of Salvadora persica, a commonly available shrub in mangrove swamps, and screening to study its bioactivity.
Another object of the invention is to isolate naturally occurring compounds from the plant Salvadora persica and identify their molecular weights, molecular formulae, melting points and their structural formulae; which will be helpful in chemical synthesis of these compounds.
Yet another object of the invention is pharmacological screening of crude extract, its fractions and purified compounds to check that the activities shown by the crude extract and fractions are maintained throughout.
Another object of the invention is to provide pharmaceutical compositions containing extracts from the plant Salvadora persica and exhibiting biological activity, especially, tocolytic activity.
The present invention seeks to overcome the drawbacks inherent in the prior art by providing the highly efficient and selective means for processing of active crude extract including fractionation, isolation and purification of the active compounds.
Furthermore, the invention provides pharmaceutical compositions containing the extract obtained from the plant Salvadora persica and useful in relieving pains in the uterine muscles.
The present invention discloses for the first time the methods of isolation, purification and pharmacological screening of all these above said commercially important compounds from a commonly available plant from mangrove swamps of the west coast of India. The said plant, identified as Salvadora persica, is an associated mangrove plant. It is a shrub, and the twigs with leaves of flowers can be hand picked. Even the crude extract and fractions exhibit therapeutic value. The important point is that the biological activity shown by the extract is maintained in the purified compounds such as methyl palmitate and betulin.
This disclosure points towards future potential clinical uses of the extract and fractions for treatment of diseases such as smooth muscle relaxant, bronchial asthma, renal colics and prevention of premature delivery. It further relates to the use of methyl palmitate and betulin in motion sickness and abdominal cramps.
The invention provides methods whereby a biologically active crude extract of an associated mangrove plant identified as Salvadora persica Linneaus 1753 is prepared. Salvadora persica Linneaus (Salvadoraceae) are shrubs or small trees with white flowers frequent in degraded mangrove swamps and saline banks all over the west coast of India and west Asia. The process disclosed in the invention further relates to the extraction, fractionation and purification of active constituent metabolites of the said plant. The invention is also concerned with the spectral identification of the compounds such as xcex2-amyrin (non-steroidalpolycyclic triterpene), betulin, ursolic acid (triterpenic acid), methyl palmitate (aliphatic ester) and lupeol (non-steroidalpolycylic triterpene). The invention also deals with molecular formulae, molecular weights, melting points and structural formulae of the said compounds. The invention provides a highly efficient and selective means for processing of active crude extract obtained from Salvadora persica, including its fractionation, isolation and purification. As used herein, the term xe2x80x9cfractionationxe2x80x9d means separating the crude extract. The terms xe2x80x9cisolationxe2x80x9d and xe2x80x9cpurificationxe2x80x9d mean separating the fractions into pure compounds.
The invention further relates to methods of screening pharmacological activities of the said compounds in mammalian tissues. The applicant has found that the crude extract obtained from the plant Salvadora persica can be separated into two fractions, i.e., chloroform and aqueous fractions. The aqueous fraction was found to exhibit tocolytic activity, which is described in detail in the present invention.
Accordingly, the invention provides a process of extracting and purifying biologically useful molecules from an associated mangrove plant which comprises the steps of:
i) collecting and processing the plant parts of Salvadora persica, 
ii) preparing a crude extract from the plant parts of Salvadora persica, 
iii) testing the crude extract using methods of pharmacology,
iv) fractionating the crude extract,
v) testing the fractions using methods of pharmacology,
vi) isolating the pure compounds by column chromatography,
vii) testing the pure compounds by using methods of pharmacology, and
viii) identifying the compounds by spectroscopy.
As said earlier, the aqueous fraction of the crude extract obtained from the mangrove plant Salvadora persica showed tocolytic activity, while the remaining 2 fractions (butanol and petroleum ether) were inactive.
The invention also provides the identification of the molecules from the spectral data. The molecular formulae of the five compounds are provided from the spectral data. The invention provides molecular weights of the molecules from EIMS. The structural formulae of the compounds are also provided from the spectral data.
Thus, a crude extract was obtained from the plant Salvadora persica. The crude extract was tested for its bioactivity and if found promising in terms of its pharmacological activity, it was fractionated using solvents with increasing polarity to obtain fractions such as petroleum ether, chloroform, butanol and aqueous fractions. Each of these were also tested for their pharmacological activity.
The five compounds purified from the extract of the plant were xcex2-amyrin (non-steroidalpolycyclic triterpene), betulin, ursolic acid (triterpenic acid), methyl palmitate (aliphatic ester) and lupeol (non-steroidalpolycyclic triterpene).
xcex2-amyrin was found to be a non-steroidalpolycyclic triterpene with the following details:
Another molecule found was betulin having:
Ursolic acid (triterpenic acid) molecule was also found in the extract. It had:
Methyl palmitate (aliphatic ester) found in the extract had:
Lupeol (non-steroidalpolycyclic triterpene) found in the extract had:
One of the compounds is betulin, which can be used in manufacturing of betulinic acid (U.S. Pat. No. 5,804,575 issued on Sep. 8, 1998). Betulinic acid has been intensively investigated as a potential therapeutic agent for a variety of diseases. Ursolic acid is added to a flavoured product to reduce aftertaste in the product and enhance its sweetness, for example, in a diet drink. It was also used as a constituent in a preparation for inhibition of skin Tumorigenesis. Extracts of some plants show vasoconstrictor and analgesic properties and also contain triterpenoid beta-amyrin. These compositions for inhibiting the formation of unwanted skin pigmentation combine high tyrosinase blocking capabilities with stability in cosmetic preparations, absence of significant cytotoxic effects and synergy of action (U.S. Pat. Nos. 5,773,014 and 5,679,393). Beta-amyrin and lupeol are used as components for dimethylsterols in medical formulations (U.S. Pat. No. 4,808,574).
Methyl palmitate is a compound used in making alcohols as mentioned in U.S. Pat. No. 6,049,013, which issued in April, 2000. Lupeol can be used as a component for several remedial medicines, insect repellents, distilleries anti tumour and chemical industries (U.S. Pat. Nos. 4,808,574; 5,962,527; and 5,908,628).
Thus, the extract of Salvadora persica contained several compounds as listed above. The aqueous fraction of the extract of the said plant Salvadora persica was then tested for its biological activity on guinea pigs. The Applicants, to their surprise, found that the aqueous fraction exhibited excellent tocolytic activity.
Accordingly, the invention provides compositions containing the aqueous fraction of the extract obtained from Salvadora persica, optionally with conventional additives for relieving pains in the uterine muscles. The composition may contain about 10 xcexcgm of the extract. Thus, the aqueous fraction of the crude extract obtained from the plant Salvadora persica is a potential tocolytic agent. The compositions may be formulated in different physical forms, as may be required. The extract may be used as such or with conventional additives, physiologically acceptable carriers, preservatives, buffers, etc., as required.
Additionally, the invention provides a method of treating tocolytic conditions, which comprises administration of therapeutically effective amount of extract obtained from Salvadora persica to the subject in need thereof. The extract may be administered at a dosage level in the range of 50 xcexcg/ml to 250 xcexcg/ml, in case of normal adults. The exact dosage will vary depending on the patient to be treated and will depend on factors such as requirements of the patient, severity of the condition being treated and the activity of the extract. The determination of optimum dosages for a particular patient is well-known to those skilled in the art.